Dynamic Distribution And Excretion Of SBDS-GPV In Ducklings

Since 2015,a novel epidemic disease(temporarily called "Duck short beak and dwarfism syndrome")with typical clinical symptoms,including weak foot,short beak(beak is shortened,and in severe case,tongue is exposed),easily fractured and severe growth retardation(so-called stiff duck),has spread in the Mule Duck and Cherry Valley Duck flock in many provinces,including Fujian,Shandong,Anhui,Jiangsu,Henan and Zhejiang,causing great economic loss to the duck industry.The pathogenic isolation and identification have demonstrated that the disease was caused by a mutant strain of Goose Parvovirus(GPV).Considering that the short beak and growth retardation were the main clinical symptoms of the disease,and to separate from its native variant,the mutant GPV strain was temporarily called "short beak and dwarfism syndrome goose parvoviruses"(SBDS-GPV).As the disease is a newly emerging infectious disease in China,a few relevant researches have been conducted.Most of the studies focus on the isolation and identification of pathogens,virus gene sequences and diagnostic PCR.However,there are few reports on the rapid immunological diagnosis.In this study,the distribution of virus in organs and tissues,as well as the excretion of virus were monitored in SBDS-GPV-infected ducklings,which provided basis for the prevention and control of the disease.1.The development of SBDS-GPV indirect immunofluorescence assay(IFA).In the detection of SBDS-GPV using IFA,anti-goose parvovirus(GPV)monoclonal antibody(Mab E16)and goat-anti-mouse Ig-FITC were used as the primary and secondary antibodies,respectively.The assay showed good specificity:it specifically recognized GPV,as indicated by a bright green fluorescence,and had no cross reactions with pathogens from other duck diseases,including MDPV,MDRV,NDRV,DHV,ATMUV and PMV.In addition to specificity,the established IFA also has the advantage of rapid,which can be beneficial for a rapid diagnosis of short beak and dwarfism syndrome(SBDS).2.The detection of dynamic distribution of SBDS-GPV in infected duck models using IFA.Two-day-old Mule ducks were infected with SBDS-GPV via oral administration and intramuscular injection.Subsequently,the distributions of virus in the organs and tissues,including heart,liver,spleen,lung,kidney,bursa,cecal tonsil,thymus,pancreas,and duodenum,were detected at different time.The results showed that both orally and intramuscularly administered viruses were distributed in the heart,liver,kidney,cecal tonsil,duodenum,and pancreas of infected ducks.In the orally administered group,the virus antigen was detected in the duodenum on the 3rd day post-infection(PI),while was mainly detected in the pancreas,duodenum,cecal tonsil,liver,and heart on the 5th day.The fluorescence intensity was strongest on the 5th to 7th day PI,and began to weaken from the 10th day while turned negative on the 14th day.In the intramuscularly injected group,the virus antigen was detected in the liver and duodenum on the 2nd day PI,while was mainly found in the liver,pancreas,spleen,duodenum,cecal tonsil,and kidney on the 3rd day.The fluorescence intensity was the strongest on the 5th to 7th day PI,and began to weaken on the 10th day while turned negative on the 14th day.The findings indicate that the organs that are most suitable for IFA in the detection of infected ducks are pancreas,liver,kidney,duodenum,heart,spleen,and cecal tonsil.3.The application of fluorescence quantitative PCR(qPCR)and virus isolation(Ⅵ)in the determination of excretion of virus in infected ducks.Two-day-old Mule Ducks were infected with SBDS-GPV,and swabs of oropharynx and cloaca were collected on days 3,5,7,10,12,14,and 21.The samples were subsequently subjected to the fluorescence quantitative PCR(qPCR)and virus isolation(Ⅵ),which can determine the excretion in infected ducks.The results were as follows:(1)the qPCR results illustrated that positive GPV fluorescence signals were detected in both oropharyngeal and cloacal samples on the 3rd day PI and the positive signal was strongest on the 5th day.Additionally,positive fluorescence signals were not detected in the oropharynx on the 12th day and in the cloaca on the 21st day.These findings suggest that the excretion time of SBDS-GPV-infected ducks through the oropharynx and cloaca are 3 to 10 d and 3 to 14 d,respectively;and(2)the Ⅵ results showed that when the cloacal samples from the 5th to 10th day PI were continuously passed to the third generation on MDEF,the cytopathic effect(CPE)was observed.In addition,IFA result was positive with strongest fluorescence signal of infected MDEF on the 5th day PI.In contrast,Ⅵ of cloacal samples from the 12th day PI was negative.These data,as a result of combined IFA and VI,indicate that the cloacal excretion time in SBDS-GPV-infected ducks is 5 to 10 d.Conclusion:SBDS-GPV is a relatively invasive virus,which can distribute through a board range of tissues and organs.Its major target organs include pancreas,liver,kidney,heart,and intestine,and it can be excreted through the oropharynx and cloaca.The excretion level in the cloaca is higher than that in the oropharynx.Thus,the excrement of SBDS-GPV-infected or dead ducks can be potentially a main source of the spread of this disease.